DE19619238A1 - Anti-adhesive properties of polyacrylic and polymethacrylic acids - Google Patents

Description

The present invention relates to polyacrylic acids and polymethacrylic acids of different molar masses for use as medicaments and in special for use in the therapy and prophylaxis of Diseases associated with excessive, mediated by selectin receptors Cell adhesion is associated with the tissue affected by the disease.

The circulation of blood cells such as B. leukocytes, neutrophils, granulocytes and monocytes is a multi-level, very complex at the molecular level Process that is only known in partial steps (Review: T. A. Springer, Cell 76, 301-314, 1994).

Recent research has shown that in immune monitoring decisive recirculation of the lymphocytes and the localization of Neutrophils and monocytes in foci of inflammation very similar molecular Mechanisms obey. So it happens with acute and chronic Inflammatory processes for the adhesion of leukocytes to endothelial cells and Emigration to the focus of inflammation and to the secondary lymphatic Organs.

Numerous specific signaling molecules such as e.g. B. Interleukins, leukotrienes and tumor necrosis factor (TNF) their G protein coupled receptors and especially tissue-specific ones Cell adhesion molecules involved, which enable a precisely controlled detection of the Ensure immune and endothelial cells. Among the most important involved in this Adhesion molecules, which are referred to below as receptors,  include the selectins (E-, P- and L-selectins), integrins and the members of the Immunoglobulin superfamily.

The three selectin receptors determine the initial phase of the Leukocyte adhesion. E-selectin is applied to endothelial cells a few hours after Stimulation, for example, by interleukin-1 (IL-1β) or tumor necrosis factor (TNF-a) expressed while P-selectin in platelets and endothelial cells is stored and after stimulation by thrombin, peroxide radicals or Substance P u. a. is presented on the cell surfaces. L-selectin will permanently expressed on leukocytes, however, becomes evident in the course of the inflammation quickly cleaved from the leukocytes.

The inflammatory processes in the early stages through selectin receptors mediated adhesion of leukocytes to endothelial cells is natural and necessary immune response to various inflammatory stimuli and injuries to the vascular tissue.

However, the course of a number of acute and chronic diseases due to the excessive adhesion of leukocytes and their infiltration into the affected tissue as well as damage to healthy tissue in the sense of an autoimmune reaction adversely affected. These include, for example Rheumatism, reperfusion injuries such as myocardial ischemia / infarction (MI), acute Pneumonia after surgery, traumatic shock and Stroke, psoriasis, dermatitis, ARDS (respiratory distress syndrome in adults) as well as those after surgical interventions (e.g. angioplasty and by-pass Operations) occurring restenosis.

The natural ligand has already been successful in animal experiments with P-selectin dependent lung injuries (M. S. Mulligan et. al., Nature 1993, 364, 149) and in myocardial reperfusion injuries (M. Buerke et. al., J.Clin.Invest. 1994, 93, 1140).  

When screening for biologically active, specific antagonists surprisingly found that (commercially, for example at the company Fluka, available) polyacrylic acids and polymethacrylic acids depending their average molecular weight, which inhibit P-selectin-mediated cell adhesion.

Accordingly, the present invention relates to a polymer of acrylic acid or methacrylic acid or its pharmacologically acceptable salts for Use as a medicine.

The polymer is preferably characterized in that it is a Polyacrylic acid, preferably its sodium salt.

Polymers of are particularly suitable for use as pharmaceuticals mentioned acids, the molar masses of 900 to 63 000 g / mol.

The polymers mentioned are particularly suitable for producing a Medicinal product for the therapy or prophylaxis of a disease associated with excessive selectin-mediated cell adhesion in that of the Disease affects tissues, preferably cardiovascular disease or a rheumatic disease.

The pharmaceuticals according to the invention are generally administered intravenously, orally or administered parenterally or as implants, but also rectal In principle, application is possible. Suitable solid or liquid galenic Preparation forms are, for example, granules, powders, tablets, coated tablets, (Micro) capsules, suppositories, syrups, emulsions, suspensions, aerosols, Drops or injectable solutions in ampoule form as well as preparations with protracted release of active ingredients, usually in their manufacture Carriers and additives and / or auxiliaries such as explosives, binders, coatings, Swelling, lubricating or lubricating agents, flavorings, sweeteners or Find solution brokers. As a commonly used carrier or  Auxiliaries are z. As magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and their derivatives, animal and vegetable oils, polyethylene glycols and Solvents such as sterile water, alcohols, glycerin and polyvalent Called alcohols.

The pharmaceutical preparations are preferably in dosage units manufactured and administered. Fixed dosage units are tablets, capsules and suppositories.

For the treatment of a patient, depending on the effectiveness of the compound, Type of application, type and severity of the disease, age and body weight of the patient, different daily doses necessary. In certain circumstances however, higher or lower daily doses may also be appropriate. The The daily dose can be administered either as a single dose single dosage unit or several small dosage units as also by multiple doses divided at certain intervals respectively. The daily dose to be administered can also depend on the number of receptors expressed during the course of the disease. It is imaginable that in the initial stages of the disease only a few receptors of the cell surface and consequently the one to be administered Daily dose is lower than in seriously ill patients.

The pharmaceuticals according to the invention are produced by the mentioned polymers with usual carrier and optionally additional and / or Bring aids in or a suitable dosage form.

The high molecular weight polyacrylic and polymethacrylic acids are very important are potent and specific P-selectin antagonists cell adhesion assays described below can be detected.  

example 1

Primary assays to study the effect on cell attachment recombinant, soluble selectin fusion proteins.

To determine the effectiveness of the polymers on the interaction between the E and P-selectins (old nomenclature ELAM-1 or GMP-140) with their ligands test, an assay is used that is only for one of these interactions is specific. The ligands are in their natural form as Surface structures offered on promyelocytic HL60 cells. Since HL60 Cells ligands and adhesion molecules of different specificity can have the desired specificity of the assay only via the Attachment partners are provided. As a binding partner were genetically engineered Soluble fusion proteins produced from the respective extracytoplasmic Domain of E or P selectin and the constant region of a human Immunoglobulin subclass IgG1 used.

Production of L-selectin IgG1

For the production of soluble L-selection-IgG1 fusion protein, the from Walz et al., 1990 published genetic construct "ELAM-Rg" used.

The plasmid DNA was expressed in COS-7 cells (ATCC) for expression DEAE-dextran transfected (molecular biological methods: see Ausubel, F. M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Struhl, K. and Smith, J.A. 1990. Current Protocols in Molecular Biology, John Wiley, New York).

The culture supernatant is obtained seven days after the transfection Centrifugation of cells and cell fragments freed and on 25 mM Hepes pH 7.0, 0.3 mM PMSF, 0.02% sodium azide and brought to + 4 ° C canceled. (Walz, G., Aruffo, A., Kolanus, W., Bevilacqua, M. and Seed, B.  1990. Recognition in ELAM-1 of the sialyl-Lex determinant on myeloid and tumor cells. Science 250, 1132-1135.)

Production of P-selectin IgG1

To produce the soluble P-selectin-IgG1 fusion protein, this is from Aruffo et al., 1991 published genetic construct "CD62Rg" used. The further procedure corresponds to the production of AI shown under L-selectin IgG1.

Aruffo, A., Kolanus, W., Walz, G., Fredman, P. and Seed, B. 1991. CD62 / -P-Selectin recognition of myeloid and tumor cell sulfatides. Cell 67, 35-44.

Production of CD4-IgG1

To produce the soluble CD4-IgG1 fusion protein, this is from Zettlemeissl et al., 1990 published genetic construct "CD4: IgG1 hinge" used. The further procedure corresponds to that shown under A1 Production of L-selectin IgG1. (Zettelmeissl, G., Gregersen, J.-P., Duport, J. M. Mehdi, S., Reiner, G. and Seed, B. 1990. Expression and characterization of human CD4: immunoglobulin fusion protein. DNA and Cell Biology 9, 347-353.).

Execution of the HL60 cell adhesion assay on recombinant, soluble Adhesion molecules

1. 96 microtiter test plates (Nunc Maxisorb) are mixed with 100 µl of one in Dilute 50 mM Tris pH 9.5 (1 + 100) goat with human IgG Antibody (Sigma) incubated for 2 hours at room temperature. After removal the antibody solution is washed once with PBS.  

2. 150 µl of the blocking buffer are in for 1 hour at room temperature leave the well. The composition of the blocking buffer is: 0.1% gelatin, 1% BSA, 5% calf serum, 0.2 mM PMSF, 0.02% Sodium azide. After removing the blocking buffer, use PBS once washed.

3. 100 μl of cell culture supernatant are added to the wells accordingly pipetted transfected and expressed COS cells. The incubation takes place for 2 hours at room temperature. After removing the Cell culture supernatant is washed once with PBS.

4. 20 µl of binding buffer are added to the wells. Of the Binding buffer has the composition: 50 mM Hepes, pH 7.5; 100 mM NaCl; 1 mg / ml BSA; 52 mM MgCl2; TmMCaCl2, 3 mM MnCl2; 0.02% sodium azide; 0.2 mM PMSF. 5 µl of the test substance are pipetted in by swirling the plate is mixed and incubated for 10 min at room temperature.

5. 50 ml of an HL60 cell culture with 200,000 cells / ml are added for 4 minutes Centrifuged 350 g. The pellet is resuspended in 10 ml RPMI 1640 and centrifuged the cells again. To mark the cells 50 µg BCECF-AM (Molecular Probes) in 5 µl anhydrous DMSO dissolved; then 1.5 ml RPMI 1640 are placed on the BCECF-AM / DMSO solution given. With this solution, the cells resuspended and incubated at 37 ° C for 30 min. After two minutes The labeled cell pellet is centrifuged at 350 g in 11 ml Binding buffer resuspended and the resuspended cells in 100 µl Distribute aliquots into the microtiter plate wells. The plate is 10 min. allowed to stand at room temperature to the bottom of the cells 30 sediment test plate. The cells have the opportunity to adhere the coated plastic.  

6. To stop the test, the microtiter plate is at a 45 ° angle completely immersed in the stop buffer (25 mM Tris, pH 7.5; 125 mM NaCl; 0.1% BSA; 2mM MgCl2; 1mM CaCl2; 3 mM MnCl2; 0.02% Sodium azide). The stop buffer is removed from the well by inversion removed and the procedure repeated two more times.

7. The measurement of the BCECF-AM marked in the wells Cells are made in a cytofluorimeter (Millipore), at one Sensitivity setting of 4, an excitation wavelength of 485/22 nm and an emission wavelength of 530/25 nm.

Results:
IC 50 values for P-selectin and for E-selectin:

Example 2

Leukocyte adhesion - testing the effectiveness of the invention Compounds in vivo (intravital microscopy in the rat).

In inflammatory processes and other conditions that activate the cytokines plays tissue destruction by immigrant or microcirculation blocking leukocytes play a crucial role. The first and for the Another critical process is the activation of Leukocytes within the bloodstream, especially in the pre- and postcapillary Area. It happens after the leukocytes stop the axial flow of blood have left to first attach the leukocytes to the Inner wall of the vessel, d. H. in the vascular endothelium. All subsequent Leukocyte effects, i. H. active migration through the vessel wall and the subsequent oriented migration in the tissue are subsequent reactions (Harlan, J.M., Leukocyte-endothelial interaction, Blood 65, 513-525, 1985).

This receptor-mediated interaction of leukocytes and endothelial cells is viewed as an initial sign of the inflammatory process. In addition to the already physiologically expressed adhesion molecules occur under the Inflammation mediators (leukotrienes, PAF) and cytokines (TNF-alpha, interleukins) for the temporally graded, massive expression of Adhesion molecules on the cells. They are currently in three groups classified: 1. immunoglobulin gene superfamily, 2. integrins and 3. selectins. During the adhesion between molecules of the Ig gene superfamily and the Protein-protein bonds expiring are in the cooperation between Selective lectin-carbohydrate bonds in the foreground (Springer, T. A., Adhesion receptors of the immune system. Nature 346, 425-434, 1990; Huges, G., Cell adhesion molecules - the key to an universal panacea, Scrips Magazine 6, 30-33, 1993; Springer, T.A., Traffic signals for lymphocyte  recirculation and leukocyte emigration; The multistep paradigm. Cell 76, 301-314, 1994).

Method:
The induced adhesion of leukocytes is quantified using an intravital microscopic examination technique in the rat mesentery (Atherton A. and Born GVR, Quantitative investigations of the adhesiveness of circulation polymorphnuclear leukocytes to blood vessel walls. J. Physiol. 222, 447-474, 1972; Seiffge , D. Methods for the investigation of the receptor-mediated interaction between leukocytes and endothelial cells in the inflammatory process, in: Replacement and supplementary methods for animal experiments in biomedical research, Schöffl, H. et al., (Ed.) Springer, 1995 (in press) ). Under inhalation ether anesthesia, permanent anesthesia is initiated by intramuscular injection of urethane (1.25 mg / kg body weight). After free preparation of vessels (femoral vein for the injection of substances and carotid artery for measuring blood pressure), catheters are integrated into them. Then the corresponding transparent tissue (mesentery) is exposed according to the standard methods known in the literature and stored on the microscope table and covered with paraffin oil at 37 ° C (Menger, MD and Lehr, H., A. Scope and perspetives of intravital microscopy-bridge over from in vitro to in vivo, Immunology Today 14, 519-522, 1993). The test substance is administered iv to the animal (10 mkg / kg). The experimental increase in blood cell adhesion is triggered by systemic administration of lipopolysaccharide (LPS, 15 mg / kg) 15 minutes after application from test substance by cytokine activation (Foster SJ, Mc Cormick LM, Ntolosi BA and Campbell D., Production of TNF -alpha by LPS-stimulated murine, rat and human blood and its pharmacological modulation, Agents and Actions 38, C77-C79, 1993, 18.01.1995). The resulting increased adhesion of leukocytes to the endothelium is quantified directly under the vital microscope or with the help of fluorescent dyes. All measurements are recorded by a video camera and saved on a video recorder. Over a period of 60 minutes, the number of rolling leukocytes (ie all visible rolling leukocytes that are slower than the flowing erythrocytes) and the number of adhering leukocytes on the endothelium (residence time longer than 5 seconds) are recorded every 10 minutes. After the end of the experiment, the anesthetized animals are euthanized without pain by systemic injection of T61 without excitation. For evaluation, the results of 8 treated and 8 untreated animals (control group) are compared (the results are given in percent).

Intravital microscopy in the rat:
a) polyacrylic acid (2'100 Na salt): dose: 3 mg / kg; Application: iv; Species: SPRD (m); Weight in g:
284 ± 9.6; Number of vessels: 15; Tube diameter in µM 28 ± 2.2; Leukocytes in 10³ / mm³: 8.2 ± 2 ,; Fibrinogen in mg / 100 ml: 96 ± 10.1; Inhibition: -3%
b) polyacrylic acid (62,900 Na salt): dose: 3 mg / kg; Application: iv; Species: SPRD (m); Weight in g:
276 ± -13.6; Number of vessels: 18; Vessel diameter in µM 27 ± 5.2; Leukocytes in 10³ / mm³: 12.5 ± 3.5; Fibrinogen in mg / 100 ml: 101 ± 12.3; Inhibition: 64%.

Claims (8)

1. Polymer of acrylic acid or methacrylic acid or their pharmacological compatible salts for use as medicines. 2. Polymer according to claim 1, characterized in that the polymer is a polyacrylic acid. 3. Polymer according to claim 2, characterized in that the polymer Sodium salt of a polyacrylic acid. 4. Polymer according to one of claims 1 to 3, characterized in that the average molecular weight of the polymer is 900 to 63,000 g / mol. 5. Use of a polymer according to one of claims 1 to 4 for Manufacture of a medicament for the therapy or prophylaxis of a Disease with excessive, through selectin receptors mediated cell adhesion in the tissue affected by the disease goes along. 6. Use according to claim 5, characterized in that the Disease is a cardiovascular disease. 7. Use according to claim 5, characterized in that the Disease is a rheumatic disease. 8. Medicament containing at least one polymer according to one of the Claims 1 to 4 and pharmaceutical excipients.